1. Field of the Invention
This invention relates to oligonucleotide probes and collections of oligonucleotide probes for detecting or localizing nucleic acid genes targets within a cell or tissue sample. In particular, the invention relates to collections of oligoprobes.
2. Background of the Invention
In situ analysis includes in situ hybridization and immunohistochemistry. In situ hybridization (ISH) employs labeled DNA or RNA probe molecules that are anti-sense to a target gene sequence or transcript to detect or localize targeted nucleic acid target genes within a cell or tissue sample. ISH has proven to be a useful tool in a number of biomedical fields, including developmental biology, cell biology, and molecular biology. ISH has been used, for example, to diagnose genetic disorders, map genes, study gene expression, and localize sites of target gene expression.
Typically, ISH is performed by exposing a cell or tissue sample immobilized on a glass slide to a labeled nucleic acid probe which is capable of specifically hybridizing to a given target gene in the cell or tissue sample (In Situ Hybridization: Medical Applications (G. R. Coulton and J. de Belleroche, eds., Kluwer Academic Publishers, 1992); In Situ Hybridization: In Neurobiology; Advances in Methodology (J. H. Eberwine, K. L. Valentino, and J. D. Barchas, eds., Oxford University Press, 1994); In Situ Hybridization: A Practical Approach (D. G. Wilkinson, ed., Oxford University Press, 1992)). The hybridization of labeled probe molecules to nucleic acids in the cell or tissue sample can then be detected using, for example, radioactive-based direct detection methods, fluorescence-based direct detection methods, or indirect detection methods based on the binding of a fluorescence-labeled protein binding to a hapten such as BrdU, digoxigenin-labeled or biotin-labeled nucleotides incorporated into probes. Hapten-based methods have been further extended to include those molecules to be bonded by binding protein-enzyme conjugates such as antibody-enzyme-conjugates and colorimetric based detection chemistry. In addition, several target genes can be simultaneously analyzed by exposing a cell or tissue sample to a plurality of nucleic acid probes that have been labeled with a plurality of different nucleic acid tags. For example, a plurality of nucleic acid probes can be labeled with a plurality of fluorescent compounds having different emission wavelengths, thereby permitting simultaneous multicolored analysis to be performed in a single step on a single target cell or tissue sample.
A significant problem associated with incorporation of labeled nucleotides into oligonucleotide probes is that the conjugation moieties that are attached to the nucleotide usually interfere with the formation of Watson-Crick base pairing, thus negatively affecting the hybridization of the probe to its target. The has been seen with use of label attached via N4-substituted cytosine nucleotides, because of steric hindrance and the expected shift to the less reactive state of a secondary amine (as seen with N4 labeled cytosine), as compared to the natural G-C bond formed with an unsubstituted cytosine (a primary amine). Any small change or interference with G-C bonding in a small oligonucleotide (25 to 50 bases) can reduce the ability of these oligos to hybridize with the intended targeted sequence.
There remains a need in the art to develop suitable probes designs for incorporating labeled nucleotides in oligonucleotide probes. We demonstrate that a few artificial sequences are viable alternatives for probe labeling and also work both singly and in complex oligonucleotide probe mixtures for detecting or localizing nucleic acid target genes within a cell or tissue sample. The development of such generic sequences and labeling strategy for probe collections has wide application in the medical, genetic, and molecular biological arts.
This interference due to labeling chemistry and hybridization stringency and kinetics is solved herein by designing the oligo to have at least two distinct functional domains, one domain or sequence to be gene specific and involved in the base pair formation, and the second domain to be an artificial, non-specific sequence (in reference to the sample's genome) comprised of spacing nucleotides and the labeled nucleotide. These elements are positioned so that these label-nucleotides are more accessible as haptens for binding proteins (immunoglobulin or avidin(s)) and thus do not interfere with Watson-Crick base pairing in the gene-specific domain.